Combined ATR and mitotic inhibition is effective against MYCN-amplified neuroblastoma.

High-risk neuroblastoma (NB) is an aggressive pediatric malignancy characterized by a high proliferative capacity, intrinsic replication stress, and poor clinical outcomes. Although mitotic inhibitors show preclinical efficacy, their clinical use is limited by dose-dependent toxicity. Here, we performed high-throughput combinatorial drug screening using MYCN-amplified human NB tumoroids to identify drugs with orthogonal mechanisms that synergize with low-dose mitotic inhibition. Promising drug combinations were prioritized using a multifactorial approach including dose-response matrices across multiple NB tumoroids and CD34⁺ cord blood cells, synergy area scores, and machine learning-based response predictions. Several therapeutic classes were selected, including kinase inhibitors, differentiation agents, and hormone therapies. Notably, combined ATR inhibition by elimusertib and mitotic KSP inhibition showed additive to mild synergy and induced NB cell death. This combination reduced tumor growth and prolonged survival in NB MYCN-amplified, chemoresistant patient-derived xenografts. Furthermore, alternating dosing schedules were well tolerated and maintained stable disease control in vivo. Collectively, these findings establish combined ATR and low-dose mitotic inhibition as a promising therapeutic strategy for MYCN-amplified NB.